Fermentation process for 9-(beta-d-arabinofuranosyl)adenine

ABSTRACT

Process for the production of 9-( Beta -Darabinofuranosyl)adenine by inoculating an aqueous nutrient medium, preferably containing sources of assimilable carbon and nitrogen and added inorganic salt and having a pH between about 6 and 8, with a 9-( Beta -D-arabinofuranosyl)adenine-producing strain of Streptomyces antibioticus, such as the strain corresponding to NRRL 3238, conducting a fermentation under aseptic aerobic conditions at a temperature between about 20* and 45* C. until a substantial quantity of 9-( Beta -Darabinofuranosyl)adenine is formed, and isolating the desired product from the fermentation mixture. The 9-( Beta -Darabinofuranosyl)adenine product of the process is useful as an antiviral agent that is active in in vitro and in vivo against both herpes and vaccinia viruses.

United States Patent Inventors John David Howells Grosse Pointe Woods;Albert Ryder, Detroit, both of Mich. Appl. No. 671,557 Filed Sept. 29,1967 Patented Oct. 26, 1971 Assignee Parke, Davis & Company Detroit,Mich.

Continuation-impart of application Ser. No. 606,044, Dec. 30, 1966, nowabandoned.

FERMENTATION PROCESS FOR 9-(B-D- ARABINOFURANOSYUADENINE 5 Claims, NoDrawings US. Cl 195/28 N, 195/80 Int. Cl C12d 13/06 Field of Search195/28 N, 80

[56] References Cited UNITED STATES PATENTS 2,757,123 7/1956 Sobin etal. 195/80 X 3,094,460 6/1963 DeBoer et a1 195/28 N PrimaryExaminer-Alvin E. Tanenholtz Altorneys- Robert R. Adams, David B.Ehrlinger, George M.

Richards and Edward J. Gall tion under aseptic aerobic conditions at atemperature between about 20 and 45 C. until a substantial quantity of9- (B-D-arabinofuranosyl)adenine is formed, and isolating the desiredproduct from the fermentation mixture. The 9-(B-D-arabinofuranosyl)adenine product of the process is useful as anantiviral agent that is active in in vitro and in vivo against bothherpes and vaccinia viruses.

JFJEIRMENTATION PROCESS FOR 9-(B-D- ARABINOFURANOSYMADENINE Thisapplication is a co ntinuatiomin-part of copending application Ser. No.606,044, filed Dec. 30, 1966, now abancloned.

SUMMARY AND DETAILED DESCRIPTION HID-CH More particularly, the inventionrelates to a fermentation process for the production of the compound ofthe foregoing formula by cultivating a selectedQ-(B-D-arabinofuranosyl)adenine-producing strain of the organismStreptomyces amibiou'cus.

In accordance with the invention, 9-(B-D-arabinofuranosyl)adenine isproduced by cultivating a selected 9-(B-D-arabinofuranosyl)adenine-producing strain of the organism Streptamycesantibioticus under artificial conditions in a suitable nutrient mediumuntil a substantial quantity of 9'(B-D- arabinofuranosyhadenine isformed. Following the period of cultivation or incubation,9-(B-D-arabinofuranosyl)adenine can be obtained from the medium byprocedures described hereinafter and can be subjected to the degree offurther purification desired. The term9-(B-D-arabinofuranosyl)adenine-producing strain of the organismStreptomyces antibiolicus, as used in the present specification andclaims, means a strain of Streptomyces antibioticus which,

described, causes the formation of a beer from which 9-(B-D-arabinofuranosyl)adenine can be obtained by the procedures set forth.

TABLE 1.MACROBCOPIC CULTURAL CHARACTERISTICS OFQ-(fi-D-ARABINOFURANOSYL) ADENINE-PRODUCING A strain of Streptomycesantibioticus suitable for the purpose of the invention has been isolatedfrom a sample of soil collected near Bosco Trecase, Naples Province,Campania, Italy. Cultures of this organism have been deposited with theUnited States Department of Agriculture, Northern Utilization Researchand Development Division, Peoria, Illinois, and are being maintained intheir permanent culture collection as N RRL 3238.

The organism is an aerobic and aeriully sporulating member of the orderActinomycetales and belongs in the genus Streptomyces as described inthe Seventh Edition of Bergeys Manual of Determinative Bacteriology(1957). Its macroscopic cultural characteristics on numerous mediauseful for identification ofmembers of this genus are shown in table 1.

When the organism is cultivated on certain agar media, the aerialmycelium is usually light to grayish yellowish brown. Cultivation inthese agar media leads to the formation of yellowish brown to moderatebrown soluble pigment that becomes reddish when the media are treatedwith sodium hydroxide. In media containing complex nitrogen sources adark brown or black soluble pigment is formed.

The spore chains are straight to flexuous, occasionally looped orloosely spiralled With age, the chains become very flexuous andirregular. The spores are smooth and elliptical to globose nd may varyin size from 0.7-1.2 microns X 0.9-1.7 microns.

In carbon utilization tests, good to fair growth was obtained with thefollowing single carbon sources: glucose, l. arabinose, D-xylose,i-inositol, D-mannitol, D-fructose, and rhamnose. Poor to fair growthwas obtained with raffinose, and poor or no growth was obtained withsucrose and cellulose.

ln micromorphology, color of aerial mycelium, and melanin production,the organism resembles S treplom yces antibioticus, and is thereforeregarded as a member of this species. In comparative laboratory studiesour organism is similar to the type culture of S. antibioticus, strainIMRU 3435. In certain respects, however, our organism is distinctlydifferent from the IMRU 3435 strain, as shown in table 2, and istherefore regarded as a new and distinct strain of S. antibiolicus, thenew strain being represented by culture number NRRL 3238.

In accordance with the invention, 9 (/3-D-arabino-furanosyl)adenine isproduced by inoculating an aqueous nutrient medium with aQ-(fi-D-arabinofuranosyl)adenineidiiih GfFiih of Strept myces omibiolicus, conducting a fermentation under aseptic aerobic conditions at atemperature between about 20 and 45 C. until a substantial quantity of9-(B-D-arabinofuranosyl)adenine is formed in the fermentation mixture,and subjecting the fermentation mixture to subsequent treatment in orderto obtain the desired product.

STRAIN OF STREPTOMYC'ES ANTIBIOTICUS CORRESPONDING TO NRRL 3238 Colorof- Reverse of substrate Culture medium Aerial myceliurn myceliumSoluble pigment Other features Yeast extract-malt extract agar Ggayishyellowish Brownish gray Moderate brown Reddish with addition of NaOH.

rown. Oatmeal agar do Lt. to moderate olive Grayishto strong D0.

rown. yellowish brown. Inorganic salts-starch agar Lt. to grayishyellow- Lt. olive gray to Lt, to moderate Do.

ish brown. moderate yellowish yellowish brown.

brown. Glycerol-esparegine agar Grayish yellowish Grayish yellow Grayishto strong Do.

brown. yellowish brown. Starch agar B Lt. to grayish yellow Lt. brownishgray to None brown. grayish yellowlsh town. 0r enic nitrate brothNitrate not reduced to nitrite- Ge atin Strong liquefaction.

Tryptonqoast extract broth Peptone-yeast extract-iron agar- Tyrosineagar Yeast extract-malt extract agar .I

Strong hydrolysis.

28 C.-good growth; 37 C.- good growth; 46 C.good growth; 50 C.-go0dgrowth.

1 Color of soluble pigment. blorn-Ltflight.

TABLE 2.COMPARISON OF il-(fl-D-ARABINOFURANOSYL)ADENINE-PRODUCING STRAINOF STREPTOMYC'ES ANTIBI OTIC'US CORRESPONDING TO NRRL 3238 WITH S.ANTIBIOTICUS CORRESPONDING TO IMRU 3435 S. antibioticus, correspondingto NRRL S. antibioticus, corresponding to IMRU 3238 3435 CharacteristicColor of aerial mycelium Light to grayish yellowish brown. Medium grayto light brownish gray. Micromor hology of aerial mycelium 1 Occasionalloops and spirals No loops or spirals observed. Soluble p gment:

Yeast extract-malt extract agar Moderate brown Grayish yellow.

Oatmeal agar Grayish to strong yellowish brow Do.

Inorganic salts-starch agar- Glycerol-asparaglne ar Eflect of NaOH on souble pigment of above media...

Tyrosine agar Dark brown.

Carbon utilization:

Sucrose Xylose... i-Iizositol- Gelatin liquefaction Milk hydrolysis...Growth on yeast ext None. D

o. Pigment unchanged. None.

I Table 1, in first five media.

For the inoculation, spores or conidia of the selected culture "ofSrreptomyces antibioricus can be used. Aqueous suspensions of the sporesor conidia containing a small amount of soap or another wetting agentcan be conveniently employed. For large fermentations it is preferableto use vigorous young aerated and agitated broth cultures of themicroorganism.

Suitable aqueous nutrient media are those containing assimilable sourcesof carbon and nitrogen and preferablyihaving a pH between about 6 and 8.Sources of carbon which are assimilable and satisfactory for use includepure carbohydrates that can be utilized by the organism as well ascommercially available carbohydrate mixtures. Some examples of thematerials that are suitable for this purpose are various sugars, such asglucose, maltose, lactose, and mannose; starch and modified starches;corn syrup; malt liquors; blacltstrap molasses; glycerol; and corn meal.The quantity of the carbohydrate present in the nutrient medium is notparticularly critical and can vary from about 0.5 to 5 percent by weightof the medium. Quantities somewhat outside of this range can also beused.

The sources of nitrogen in the nutrient medium can be of an organic,inorganic, or mixed organic-inorganic nature. Some examples of the manynitrogenous substances that can be employed in the nutrient medium areamino acids, peptones, hydrolyzed and unhydrolyzed proteins, fish meal,soybean meal, peanut meal, cottonseed meal, wheat gluten, corn steepliquor, dehydrated corn steep liquor, meat extracts, inorganic nitrates,urea, and ammonium salts. Because of the crude nature of most of thereadily available nitrogen sources, the quantity to be added to themedium varies according to the purity, and it is not readily possible tospecify a definite quantity of nitrogenous source material that shouldbe added to the medium. It can be said, however, that, for practicalpurposes, nitrogenous materials need not exceed 6 percent by weight ofthe total fermentation medium nd can be present in a considerably loweramount.

The presence ofa certain amount of mineral salts and traces of growthfactors of unknown composition is desirable in order to obtain the bestyields of 9-(l3-D-arabinofuranosyl)adenine. Many readily available crudematerials, such as corn steep liquor, yeast preparations, soybean oilmeal, molasses fermentation residues, and other products of likecharacter contain such inorganic salts and growth factors, and theinclusion of one or more of these materials in the fermentation mediumis desirable. In order to ensure the presence of adequate amounts of themineral components of the medium, it is also advantageous in many casesto add some inorganic salts, such as sodium chloride, sodiumbicarbonate, potassium phosphate, sodium acetate, calcium carbonate, andmagnesium sulfate, as well as trace quantities of minerals such as r a.s assesses? sasas efl rrfim:

centration of a given mineral salt is between 0.1 and 1 percent byweight of the nutrient medium.

The cultivation of the selected strain of Streptomyces an ribioticus inthe aqueous nutrient medium can be carried out in a number of differentways. For example, the organism can be cultivated under aerobicconditions on the surface of the medium; or it can be cultivated beneaththe surface of the medium, that is, in the submerged condition, providedthat an adequate supply of oxygen is furnished.

The preferred method for producing Q-(B-D-arabino-furanosyl)adenine on alarge scale is by the fermentation of a 9-(fl-D-arabinofuranosyl)adenine-producing strain of Streptomycesantibioticus in a submerged or deep culture. According to thisembodiment of the invention, a sterile aqueous nutrient m iu iiwlated wl i essl c isulsureaa ncu ted with agitation and aeration under asepticconditions at a temperature between about 20 and 45 C., preferably inthe neighborhood of 33-40 C., until a substantial quantity of 9-(B-D-arabinofuranosyl)adenine is found in the culture liquid. The lengthof time required for the maximum yield varies with the size and type ofequipment used, the rates of agitation and aeration, the specificorganism culture and other factors. in large scale commercialfermentations carried out in the tanktype fermentors, maximum productionis usually reached in about 3 to 7 days. Shorter fermentation periodscan also be used, but usually produce a lower yield. When thefermentation is carried out in shaken flasks, the time required formaximum production may be somewhat longer than when large fermentationtanks are used.

Under the submerged culture conditions, the microorganism develops asrelatively discrete particles dispersed throughout the nutrient mediumin contrast to the relatively continuous pellicle present on the surfaceof the medium in the surface culture method. By virtue of thisdistribution of the organism throughout the medium, large volumes of theinoculated nutrient medium can be used in the cultivation of the organism in the tanks and vats customarily employed in the fermentationindustry. Stationary vat fermentors equipped with agitation and aerationdevices are particularly suitable for large-scale production, althoughfermentation equipment for other designs can also be used. For theproduction of smaller quantities of product or for the preparation ofcultures of the organism to be used as inoculum for large-scalefermentations, the submerged culture method can be carried out in smallflasks or jars which are either shaken or stirred by suitable mechanicalmeans.

in the submerged culture method, agitation and aeration of the culturemixture can be accomplished in a number of ways. Agitation can beprovided by turbines, paddles, impellers or other mechanical agitationdevices, by revolving or shaking the fermentor itself, by variouspumping devices or by the passage of air or oxygen through the medium.Aeration can be brought about by injecting air or oxygen into thefermentation mixture through open pipes, perforated pipes, or pipescontaining a porous diffusion section; or it can be brought about byspraying, splashing or spilling the medium into or through anoxygen-containing atmosphere.

An alternative to the preferred submerged culture method is the surfaceculture method of producing 9-(B-D-arabinofuranosyl)adenine according towhich a shallow layer, usually less than 2 cm., ofa sterile, aqueousnutrient medium is inoculated with a9-(B-D-arabinofuranosyl)adenine-producing strain of Streptamycesantibioticur and the inoculated mixture is incubated under aerobicconditions at a temperature between about and 45 C. The product is thenobtained in a manner similar to that described for the submerged culturemethod.

Upon completion of the fermentation phase of the process, the desiredproduct can be obtained in a number of ways. In the case of thesubmerged culture method, the preferred method is as follows. Themycelium is separated by such means as filtration or centrifugation. Thefilter cake is washed well with water, the washings are combined withthe filtered beer, and the combined liquids are concentrated underreduced pressure to about one-twelfth the original volume. Theconcentrated solution is cooled at about 5 C. for an extended period(from several hours to several days, depending on the volume), and thesolid that precipitates is isolated by filtration with the aid ofdiatomaceous earth. The filter cake is then extracted well with boilingwater, and the combined extracts are cooled at about 5 C. untilprecipitation is complete. The crystalline9-(B-D-arabinofuranosyl)adenine that precipitates is isolated byfiltration and further purified by successive crystallizations fromboiling water.

Alternatively, the desired product can be obtained from the submergedculture method by employing adsorption techniques as follows. Again,after fermentation is complete, the mycelium is separated by such meansas filtration or centrifugation. The crude pidd uct isthen adsorbedbytating the filtered beer with activated charcoal, or other adsorbingagent. The adsorption can be carried out either in batches or bycontinuous flow through an adsorption column. In the preferred batchmethod, from 0.1 to 0.6 percent, preferably 0.35 to 0.4 percent,weight/volume, of the preferred charcoal adsorbent is added to thefiltered beer, and the resulting mixture is stirred for 1-3 hours. Insome cases, it may be desirable to remove impurities from the filteredbeer prior to charcoal treatment either by extracting it with animmiscible organic solvent, such as ethylene dichloride or ethylacetate, or by treating it with a synthetic cation exchange resin in thesodium form. The crude product is isolated by eluting the charcoaladsorbent with aqueous acetone or with an aqueous lower alkanol andevaporating the eluate under reduced pressure. The solid residueobtained is then purified either by successive crystallizations fromwater or a lower alkanol or by the following procedure. The solidresidue is extracted with a water-immiscible liquid alkanol, such asn-butyl alcohol, and the extract is poured onto a column of alumina (pH5-6). The alumina column is eluted with 95 percent aqueous ethanol, theeluate is evaporated, and the solid obtained is crystallized from wateror a lower alkanol to give the desired 9-(B-D- arabinofuranosyl)adenine.

The product of the process of the invention, 9-(B-D-arabinofuranosyl)adenine, is useful as an antiviral agent that is activein vitro and in vivo against both herpes and vaccinia viruses.

The invention is illustrated by the following examples.

EXAMPLE 1 Sterile agar slants are prepared using the Slreptomycessporulation medium of Hickey and Tresner [R. J. Hickey and H. D.Tresner, J. Bact., Vol. 64, pages 891-892 (1952)]. Four of these slantsare inoculated with lyophilized spores of Streptomyces antibioticuscorresponding to NRRL 3238, incubated at 28 C. for 7 days or u ntilaerial spore growth is well advanced, and then stored at 5 C. The sporesfrom the four slants are suspended in 40 ml. of 0.1 percent sterilesodium heptadecyl sulfate solution.

A nutrient medium having the following composition is prepared:

Glucose monohydrate 2.0% Soybean meal, solvent extracted,

44% protein 1.0% Animal peptone (Wilson's Protopeptone I59) 0.5%Ammonium chloride 0.2% Sodium chloride 0.5% Calcium carbonate 0.25%

Water to make 100% The pH of the medium is adjusted with l0-normalsodium hydroxide solution to pH 7.5.

12 liters of this medium is placed in a 30-liter stainless steelfermentor. The medium is sterilized by heating it at 121 C. for minutes,allowed to cool, inoculated with the 40-ml. spore suspension describedabove, and incubated at 25-27 C. for 32 hours while being agitated at200 rpm. with air being supplied at the rate of 12 liters per minute.About 38 g. ofa mixture of lard and mineral oils containing monoanddiglycerides is added in portions during this time to prevent excessivefoaming.

Sixteen liters of a nutrient medium having the composition describedabove is placed in each of four 30-liter stainless steel fermentors. ThepH of the medium. in each fermentor is adjusted with lO-normal sodiumhydroxide solution to pH 7.5, and each is sterilized by heating at 121C. for 90 minutes. Upon cooling, the medium in each fermentor isinoculated with 800 ml. of the fermentation mixture described above, andeach is incubated at 25-27 C. for 96 hours while being agitated at 200r.p.m. with air being supplied at the rate of l6 liters/minute. About170 g. of the antifoam mixture described above is added in portionsduring this time to the iriedium in each fermentor.

The fermentation mixtures from the four fermentors are combined andfiltered with the aid of diatomaceous earth. A material such as Celite545 can be used. The filtrate is concentrated under reduced pressure toa volume of 10 liters, and the concentrate is treated with 200 g. ofactivated charcoal (for example, Darco 6-60), stirred at roomtemperature for one hour, and filtered. The charcoal cake is washed with7.5 liters of water, and then extracted with three e l O-liter portionsof 50 percent aqueous acetone. The three aqueous acetone extracts arecombined, concentrated under reduced pressure to approximately oneliter, and chilled at 5 'C. for 48 hours. The solid9-(B-D-arabinofuranosyl)adenine that precipitates is isolated andpurified by successive crystallizations from boiling methanol and fromboiling water; m.p. 262-263 C.

In the foregoing procedure, when the temperature of incubation in thetwo fermentation stages is raised from 2S-27 C. to 36-38 C., the sameQ-(B-D-arabinofuranosyl)adenine product is obtained in higher yields.

EXAMPLE 2 A nutrient medium having the following composition isprepared:

Glucose monohydrate 2.0 7: Soybean meal, solvent extracted.

44% protein 1.0% Animal peptonc (Wilsons Protopeptone 159) 0.5% Ammoniumchloride 0.2%

Sodium chloride 0.5%

Calcium carbonate 0.25%

Water to make The pH of the medium is adjusted with IO-normal sodiumhydroxide solution to pH 7.5.

10 gallons of this medium is placed in a 30-gallon stainless steelfermentor. The medium is sterilized by heating it at 121 C. for 60minutes, allowed to cool, and inoculated with a 40- ml. spore suspensionprepared as described in the first paragraph of example 1 above. Anantifoam mixture (150 ml.) consisting of lard and mineral oilscontaining monoand diglycerides (for example, Swift's No. 51 inedibleDefoamer) is added and the mixture is incubated at 26-27 C. for 59 hourswith aeration at a rate of 6.25 cubic feet per minute.

Three hundred gallons of a nutrient medium having the compositiondescribed above is placed in a 500-gallon lnconel-clad fermentor. Themedium is adjusted to pH 7.5 with 10-normal aqueous sodium hydroxide andthen sterilized by' heating it at 121 C. for 30 minutes. Upon cooling,the medium is inoculated with 10 gallons of the fermentation mixturedescribed above, about 1,000 ml. of the antifoam mixture described aboveis added, and the mixture is incubated at 2425.5 C. for 24 hours withagitation at 84 r.p.m. and aeration at a rate of 45 cubic feet perminute. During this period an additional 5 13 ml. of antifoam is addedin portions.

1,200 gallons of a nutrient medium having the same composition describedabove is placed in each of two 2,000-gallon lnconel-clad fermentors. Themedium in each fermentor is adjusted to pH 7.5 with -normal aqueoussodium hydroxide and sterilized by heating at 121 C. for 30 minutes.Upon cooling, each is inoculated with 150 gallons of the fermentationmixture described in the preceding paragraph, 10 liters of antifoam isadded to each, and each is incubated at 24.5-26.5 C. for 95 hours withagitation at 125 r.p.m. and aeration at a rate of 120 cubic feet perminute. During this period an additional 40-46 liters of antifoam isadded on demand.

The fermentation mixtures from the two fermentors are combined, adjustedto pH 7.2 with 10-normal aqueous sodium hydroxide, slurried with 300pounds of diatomaceous earth, and filtered. The filtrate is concentratedunder reduced pressure to a volume of 500 gallons, and the concentrateis treated with 87 pounds of activated charcoal, stirred at roomtemperature for one hour and filtered. The charcoal cake is washed with400 gallons of water, and then extracted with three SOD-gallon portionsof 50 percent aqueous acetone. The three aqueous acetone extracts arecombined, concentrated under reduced pressure to a volume of 45 gallons,and chilled at 5 C. for 48 hours. The solid9-(B-D-arabinofuranosyl)adenine that precipitates is isolated andcrystallized successively from methanol and from water; m.p. 262-263 C.

In the foregoing procedure, improved yields of the 9-(fl-D-arabinofuranosyl)adenine product are obtained when the incubationtemperature is raised in the first fermentation stage from 2627 C. to29-30 C., in the second fermentation stage from 2425.5 C. to 29-30 C.,and in the final fermentation stage from 24.5-26.5 C. to 363 8 C.

EXAMPLE 3 A nutrient medium having the following composition isprepared:

Glucose monohydrate 2.0%

Soybean meal, solvent extracted,

(Wilson's Protopeptone I59) 0.5% Ammonium chloride 0.2% Sodium chloride0.5% Calcium carbonate 0.25%

Water to make 100% The pH of the medium is adjusted with IO-normalsodium hydroxide solution to pH 7.5.

12 liters of this medium is placed in a 30-liter stainless steelfermentor. The medium is sterilized by heating it at 121 C. for 90minutes, it is allowed to cool, and is inoculated with a 40- ml. sporesuspension prepared as described in the first paragraph of example 1above. The inoculated medium is then incubated at 2930 C. For 36 hourswhile being agitated at 200 r.p.m. with aeration being supplied at therate of 12 liters per minute. About 44 g. of mixture of lard and mineraloils containing monoand diglycerides is added in portions during thistime to prevent excessive foaming.

A nutrient medium having the following composition is next prepared:

Glucose monohydrate 2.0% py mea solvent xt a t?! 44% protein 10% Sodiumchloride 05% Calcium carbonate 0.25%

Water to make The pH of the medium is adjusted with l0-normal sodiumhydroxide solution to pH 7.5.

10 gallons of his second medium is placed in a 30-gallon stainless steelfermentor. The medium is sterilized by heating it at 121 C. for 60minutes, it is allowed to cool, and is inoculated with about 400 ml. ofthe fermentation mixture described above. An antifoam mixture 150 ml.)consisting of lard and mineral oils containing monoand diglycerides (forexample, Swift's No. 51 Inedible Defoamer) is added, and the mixture isincubated at 29-30 C. for 24 hours with aeration at a rate of 6.3 cubicfeet per minute.

300 gallons of nutrient medium having the same composition as theIO-gallon seed medium described above is placed in SOO-gallonlnconel-clad fermentor. The medium is adjusted to pH 7.5 with lO-normalaqueous sodium hydroxide and then sterilized by heating it at 121 C. for30 minutes. Upon cooling, the medium is inoculated with 10 gallons ofthe fermentation mixture described immediately above, about 1,000 ml. ofthe antifoam mixture described above is added, and the mixture isincubated at 303 1 C. for 24 hours with agitation at 84 r.p.m. andaeration at a rate of 45 cubic feet per minute. During this period, anadditional 4,000 ml. of antifoam is added in portions.

1,200 gallons of a nutrient medium having the same composition as thel0-gallon and 300-gallon media described above is placed in each of two2,000 g allon hi cdnel clama mentors. The medium in each fermentor isadjusted to pH 7.5 with l0-normal aqueous sodium hydroxide andsterilized by heating at 121 C. for 30 minutes. Upon cooling, each isinoculated with 150 gallons of the fermentation mixture described in thepreceding paragraph, 10 liters of antifoam is added to each, and each isincubated at 36.5-38 C. for 144 hours with agitation at 125 r.p.m. andaeration at a rate of 120 cubic feet per minute. During this period, anadditional -196 liters of antifoam are added on demand.

The fermentation mixtures from the two fermentors are combined, filteredthrough a plate and frame press with the aid of diatomaceous earth, andthe filter cake is washed with 100 gallons of water. The filtrate andwash water are combined, the combined liquids are concentrated underreduced pressure to about one-twelfth the original volume, and theconcentrated solution is cooled at 5 C. for 72 hours. The solid thatprecipitates from the cooled solution is isolated by filtration with theaid of diatomaceous earth, the filter cake is extracted twice withboiling water, first with 28 gallons and then with 40 gallons, and thecombined aqueous extracts are cooled at 5 C. for 2 hours. Thecrystalline 9-(B-D-arabin0furaricTsfifa denine that precipitates fromthe cooled solution is isolated by filtration and purified bycrystallization twice from boiling water, the first crystallization from28 gallons and the second from 20 gallons of water; m.p. 262-263 C.

We claim:

1. Process for the production of 9-(B-D-arabinofuranosyl)adenine whichcomprises inoculating an aqueous nutrient medium with a9-(,B-D-arabinofuranosyl)adenineproducing strain of Streptomycesantibioticur, incubating the inoculated medium at a temperature betweenabout 20 and 45 C. under aerobic conditions, and isolating the 9-(B-D-arabinofuranosyl)adenine product.

nutrient medium as a pH between 6 and 8.

3. Process according to claim 1 wherein the aqueous nutrient mediumcontains sources of assimilable carbon and nitrogen and added inorganicsalt.

4. Process according to claim 1 wherein the inoculated medium isincubated at a temperature between 33 and 40 C.

5. Process according to claim 1 wherein the inoculated medium isincubated under submerged culture conditions.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent N D t dOCtCbET 26,

John David Howells et a1. Inventor(s) It is certified that error appearsin the above-identified patent and that said Letters Patent are herebycorrected as shown below:

Column 8, line 61, amend "2" to read 72 last line, amend "as" to readhas Signed and sealed this 2nd day of May 1972.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. ROBERT GOTTSCHALK Attesting Officer Commissionerof Patents M P010 0 {10 6a) USCOMM-DC 60376-P69 W U 5. GOVERNMENTPRINTING OFFICE 1969 0-365-334

2. Process according to claim 1 wherein the aqueous nutrient medium as apH between 6 and
 8. 3. Process according to claim 1 wherein the aqueousnutrient medium contains sources of assimilable carbon and nitrogen andadded inorganic salt.
 4. Process according to claim 1 wherein theinoculated medium is incubated at a temperature between 33* and 40* C.5. Process according to claim 1 wherein the inoculated medium isincubated under submerged culture conditions.